Coupling factor ATPase complex of Rhodospirillum rubrum. Purification and properties of a reconstitutively active single subunit.

Extraction ofRhodospirillum rubrum chromatophores by LiCl in the presence of ATP results in more than 95% loss of both ATP synthesis and hydrolysis activities. The lost activities are completely restored when the LiCl-ATP extract is recoupled to the LiCl-depleted membranes. The factor present in this LiCl-ATP extract, has been purified by ammonium sulfate precipitation and by gel filtration through Sephadex G-ZOO columns. The specific recoupling activity of the protein LiCl factor increased by Sl-fold during the purification steps. This purified LiCl factor consists of one subunit, which migrates on sodium dodecyl sulfate gels parallel to the /3 polypeptide of the R. rubrum coupling factor, and very near to the p polypeptide of the chloroplast coupling factor. The molecular weight of this subunit is 55,500, as measured by sodium dodecyl sulfate gel electrophoresis and equilibrium sedimentation pattern in the analytical ultracentrifuge. The purified single subunit does not possess any ATPase activity even after activation treatment. However, its reattachment to the LiCl-depleted membranes results in reconstitution of both photophosphorylation and ATPase activities. It is, therefore, concluded that LiCl extraction releases only the /3 subunit from R. rubrum chromatophores, leaving all the other coupling factor subunits still attached to the membrane. The results also indicate that, although the solubilized P-subunit does not catalyze ATP hydrolysis, the membrane-bound ATP synthesis and hydrolysis are strictly dependent on the presence of this subunit.